The cell wall envelope of Gram-positive bacteria can be thought of as a microbial organelle with anchored surface proteins that require specific signals and targeting mechanism for their assembly. Previous work identified sortases, enzymes that recognize short peptide sequences within sorting signals of secreted proteins. Upon cleavage of a protein substrate immediately following a conserved threonine residue, sortases form a thioester acyl intermediate between the C-terminal carboxyl group of the substrate and their active site cysteine residue. This intermediate is subsequently resolved by the nucleophilic attack of an amino group, which completes the transpeptidation reaction and restores the active site of sortase. Four classes of sortases are distinguished on the basis of sequence homology and enzyme function. One of these, sortase A, cleaves all surface proteins harboring LPXTG sorting signals and tethers its products to the amino group of lipid II, the precursor of cell wall biosynthesis. In contrast, sortase D cleaves only the sorting signals of pilin subunits. By accepting the side chain amino group of lysine (K) within the YPKN motif of pilin precursors, sortase D assembles pili on the surface of Gram-positive bacteria. Bacillus cereus pili are formed from two subunits, the tip protein (BcpB) and the major pilin (BcpA), which generates the pilus shaft. Sortases A and D collaborate on the topic of pilus formation; their assembled product is immobilized in the cell wall and extends 0.3-1.5 <m onto the bacterial surface. Here we address the molecular mechanisms that enable sortases A and D to form pili and explore the virulence attributes of these structures towards the establishment of anthrax-like disease. Other work aims to characterize the genes and mechanisms that enable sortase A to anchor surface proteins at unique sites with the cell wall envelope of Staphylococcus aureus, processes that are governed by two distinct types of signal peptides. Chemical genetic studies seek to characterize small molecule inhibitors that block enzyme function for any one or all of the four sortases and to test their attributes in disrupting the pathogenesis of infections caused by Staphylococcus aureus and Bacillus cereus. Sortases are essential for the pathogenesis of many human or animal infections caused by Gram-positive bacteria. Appreciation of their molecular mechanisms and the structural as well as chemical nature of their inhibition will aid in the development of small molecules with therapeutic value for the treatment of bacterial infections. PUBLIC HEALTH RELEVANCE: Gram-positive bacteria cause many different human diseases and the morbidity and mortality of these infections, largely due to the development of antibiotic resistance traits, continues to increase in the United States. By studying the molecular mechanisms whereby Gram-positive bacteria promote the anchoring and surface display of proteins that enable the pathogenesis of human infections, we aim to increase knowledge about infectious processes. By developing inhibitors that prevent the assembly of proteins in the bacterial envelope we aim to develop antiinfectives that may be useful for the treatment of human infections.